PROJECT SUMMARY/ABSTRACT At birth, the alveolar epithelium must mitigate effects of oxidative stress, combat inhaled microorganisms and modulate the immune environment to protect itself from damaging inflammation. How does this occur? We postulate that the type II cell serves a crucial role through production of surfactant and immune modulators. In preterm infants, decreased surfactant and exacerbated inflammation can impair alveolar development and result in bronchopulmonary dysplasia (BPD), a chronic lung disease with significant morbidity and mortality. The major surfactant protein, SP-A, an immune modulator, is developmentally upregulated in fetal lung with type II cell differentiation and surfactant phospholipid synthesis. SP-A expression and type II cell differentiation in cultured human fetal lung (HFL) epithelial cells are stimulated by cAMP and inhibited by TGF-b and hypoxia. Mechanisms for O2?dependent induction of type II cell differentiation and SFTPA expression are not fully understood. Recently, we discovered that the redox-regulated transcription factor, NRF2 and its co-regulated target genes, C/EBPb and PPARg, were markedly induced by cAMP in HFL type II cells in an O2-dependent manner. In mouse fetal lung (MFL), a developmental increase in Nrf2, C/ebpb and Pparg, and a decrease in the Nrf2 inhibitor Keap1 were observed between 14.5 and 19.5 (term) days post-coitum (dpc), with temporal induction of SP-A and immune modulators, NADH:quinoneoxidoreductase 1 (NQO1), tryptophan 2,3-dioxygen- ase (TDO2, which catalyzes kynurenine synthesis), and the kynurenine receptor, AhR. Nrf2 KO mice manifest persistent lung inflammation and exacerbated injury in response to sublethal hyperoxia. Notably, miR-29 family members, which are induced by Nrf2 and directly target Nrf2 inhibitors Keap1 and TGF-b, are upregulated with type II cell differentiation. NRF2 binds to response elements in promoters of anti-oxidant and immunomodu- latory genes. We recently found that NQO1, TDO2, AhR, were upregulated by NRF2 in HFL and MFL epithelial cells during differentiation in culture. In studies outlined in the following Specific Aims, we will use cultured human NSCLC adenocarcinoma (adenoCa) cell lines and MFL epithelial cells, wild-type (WT) and gene- targeted male and female fetal and neonatal mice to test the novel hypothesis that NRF2, acting with C/EBPb and PPARg, serves a crucial role in fetal and neonatal lung by promoting type II cell differentiation and enhancing production of key immune modulators that alter the immune cell environment to protect the perinatal alveolar epithelium from oxidative and inflammatory stress. The following Specific Aims are proposed: (1) use cultured human adenoCa cells, MFL epithelial cells and miR-29 KO mice to define the regulatory networks of NRF2, C/EBP?, PPARg and miR-29, their roles in type II cell differentiation and expression of immune modulators; (2) analyze effects of a type II cell-specific deletion of Nrf2 in mice on expression of key transcrip- tion and immune modulators, and on lung development and pathogenesis after hyperoxia exposure; (3) assess the roles of NRF2 and SP-A on the composition and regulation of immune cells within fetal and postnatal lung.